Voltage-sensing domains (VSDs) confer voltage dependence onto the effector domains of membrane proteins. In the classical Na+, K+ and Ca++ channels that generate the action potential and control neural secretion and muscle contraction, four VSDs work in concert to control gates in a pore located at the interface between the pore domains of the four subunits. During the previous grant cycle we discovered that the situation is different in two recently isolated proteins: the sea squirt voltage-sensitive phosphatase, Ci-VSP and the human voltage- gated proton channel, Hv1. Both proteins have homologues across phyla, including in vertebrates. The VSPs provide a previously missing connection between membrane excitation and ion transport by PI(4,5)P2-sensitive channels and pumps, while the Hv channels play an important role in phagocytosis. We developed a new method of single molecule microscopy that enabled us to count the subunits of these proteins and combined these with electrophysiology to count pores and voltage clamp fluorometry to detect structural dynamics. We found that the two new members of the family break the tetrameric mold, with Ci-VSP being a monomer and Hv1 a dimer. In Hv1 we have shown that each subunit of the dimer has its own pore, voltage sensor and gate and we find now that, although the subunits can function on their own, they gate cooperatively in the dimer. We propose to determine how a single ion channel domain can combine sensor and effector functions that are separate in other channels and to understand the structural basis of the cooperativity. The work will provide insight into how a modular domain like a VSD has been shuffled around in evolution to confer voltage dependence onto a variety of membrane-associated proteins. PUBLIC HEALTH RELEVANCE: The proposed work will provide insights into the mechanism of function of voltage-gated ion channels that that generate the action potential and control neural secretion and muscle contraction, on VSPs, which provide a poorly understood modulatory signal by which membrane excitation can feed back on ion transport, and the proton channels that play a key role in killing bacteria during phagocytosis.